小桐子快速繁殖方法 Jatropha rapid propagation method

技术领域 Technical Field

[0001] 本发明涉及生物组培育苗技术领域，尤其涉及一种小桐子快速繁殖方法。 [0001] The present invention relates to the field of biological plant tissue culture technology, particularly to a method for rapid propagation of Jatropha. 背景技术 Background

[0002] 小桐子(/airoMa curcas L )，又叫麻疯树、麻风树、膏桐、黑皂树、木花生、油芦子、亮桐、臭梧桐等，属大戟科(^Modiaceae)麻疯树属ijatropha L )，落叶灌木或小乔木，是一种多年生木本油料植物。 [0002] Jatropha (/ airoMa curcas L), also known as jatropha, jatropha curcas, black soap tree, wood peanut oil Lu son, Liang Tong, and other smelly Indus, Euphorbiaceae (^ Modiaceae) Jatropha ijatropha L), deciduous shrub or small tree, is a perennial woody plant. 小桐子原产美洲，广泛分布于热带亚热带地区。 Jatropha American origin, widely distributed in tropical and subtropical regions.

[0003] 小桐子可全株开发，其果实、枝、叶均能利用。 [0003] The whole plant Jatropha can be developed, the fruit, branches, leaves can use. 小桐子种子含油率高，经过加工可制成生物柴油。 Jatropha seed oil is high, after processing can be made into biodiesel. 小桐子种子、树皮、叶、根和乳汁中含有多种成分的生物药源，可提取制作生物医药和生物农药。 Jatropha seeds, bark, leaves, roots, and milk contain a variety of components of biological medicine source, can be extracted make biomedical and bio-pesticides. 小桐子种子加工后的油饼蛋白质含量较高，脱毒后可制作生物饲料，未脱毒的可制作优质的有机生物肥。 Jatropha seed cake of high protein content of processed, can produce bio-feed after detoxification, detoxification can not make high-quality organic fertilizer. 另外，小桐子茎叶有毒，牲畜不吃，病虫较少，不易燃烧， 可作为田间地边的生物篱和防风防火屏障。 In addition, Jatropha stem toxic, livestock eat fewer pests, not combustion, can be used as bio-field fence and wind to the edge of the fire barrier. 培育小桐子能源林，利用其种子提炼生物柴油是小桐子产业发展的主要方向。 Cultivation of Jatropha forest, with its seed extract biodiesel Jatropha industry is the main direction of development.

[0004] 我国现有栽培和半野生的小桐子，其繁殖主要靠种子繁殖和扦插繁殖，繁殖周期长、成本高。 [0004] our existing cultivated and semi-wild Jatropha, which depends mainly on seed multiplication and reproduction cutting propagation, long reproductive cycle, high cost. 又因其为木本植物，采用常规育种来改变其遗传性状相当困难，因此采用现代生物技术如转基因技术等成为首选。 Because of its woody plants using conventional breeding to alter their genetic traits quite difficult, so the use of modern biotechnology such as transgenic technology have become the first choice. 一些国家和地区已经开始了对小桐子进行了分子水平的育种工作。 Some countries and regions have begun to carry out the molecular level Jatropha breeding work. 选育的优良品种大量繁殖，通过组培方式快速繁殖小桐子将在小桐子选育种生产上有重要的应用价值。 Breeding of fine varieties thrive, by way of tissue culture and rapid propagation of Jatropha will have important applications in breeding of Jatropha production.

[0005] 目前，关于小桐子的组织培养及快速繁殖的报道较多，例如：陆伟达、魏琴、唐琳等发表的《麻疯树愈伤组织的诱导及快速繁殖》（《应用与环境生物学报》，2003，9 (2)： 127^130.)以及陈金洪、高敏、黄记生发表的《麻疯树茎段离体培养及快速繁殖研究》（《广西农业科学》，2006,37 (3):22广223.)等论文均涉及小桐子的组织培养及快速繁殖技术。 [0005] Currently, tissue culture and rapid propagation of Jatropha reports of more, for example: Lu Wei Da, Wei Qin, Tang Lin et published "Jatropha induce callus and Rapid Propagation" ("Application and Environmental Biotechnology University ", 2003,9 (2): 127 ^ 130) and Chen Jinhong, Gao Min, Huang Jisheng published" Jatropha stem segments in vitro culture and rapid propagation of "(" Guangxi Agricultural Sciences ", 2006,37 (3) : 22 223. Canton) and other papers involve Jatropha tissue culture and rapid propagation techniques. 但是，现有技术存在以下不足： However, the prior art has the following disadvantages:

1、均采用不同的激素浓度组合诱导愈伤组织和芽分化，未经过再生芽增殖，其增殖速率低； 1, are used in different concentrations and combinations of hormones to induce callus buds, not through regeneration bud proliferation, the proliferation rate is low;

2、均采用不同外植体诱导愈伤组织，其不同外植体诱导频率相差及大。 2, are used in different explants callus, its different frequency difference Iduced and large.

[0006] 采用上述方法不能达到工厂化生产的需要，另有研究者进行了腋芽快繁研究，例如：李化、曾妮、贾勇炯等人发表的《麻疯树的促腋芽分枝快繁及生根诱导》（《四川大学学报：自然科学版》，2006，43 (5):1116〜1120.)，所用外植体为种子萌发的胚芽来诱导增殖。 [0006] The above method does not achieve the desired factory production, while researchers performed rapid propagation of axillary buds, such as: promoting Axillary branches of propagation and Lee, who Ni, 贾勇炯 et al.'s "Jatropha rooting "(" Journal of Sichuan University: Natural Science ", 2006,43 (5): 1116~1120), the proliferation induced by the explants seed germination germ. 但尚未见公开采用再生芽来诱导增殖的繁殖技术。 But it has not been disclosed to the use of renewable bud proliferation induced breeding technology.

发明内容 DISCLOSURE

[0007] 本发明实施例所要解决的技术问题在于，提供一种小桐子快速繁殖方法，以通过组织培养大规模高效地生产小桐子。 Technical Problem [0007] embodiment of the present invention to be solved is to provide a method for rapid propagation of Jatropha to scale efficiently through tissue culture production of Jatropha.

[0008] 为解决上述技术问题，本发明提供如下技术方案：一种小桐子快速繁殖方法，包括如下步骤：准备再生芽步骤，以小桐子成熟种子为材料，经培育获得无菌再生芽； 增殖诱导步骤，将再生芽切下，接入增殖培养基进行增殖培养，从而获得增殖的丛芽； 伸长培养步骤，将增殖后的丛芽切下，接入伸长培养基进而获得茎叶伸长的均勻的 [0008] In order to solve the above problems, the present invention provides the following technical solutions: one Jatropha rapid propagation method comprising the steps of: preparing a regeneration bud steps to mature Jatropha seed material obtained by cultivating sterile regeneration bud; Proliferation inducing step, the regenerated shoots cut down access to proliferate proliferation culture medium to obtain proliferation of buds; elongation incubation step, the next cut buds proliferated, the access elongation medium and then get stem extension long uniform

芽； Bud;

壮苗培养步骤，将伸长后的芽接入壮苗培养基进行壮苗培养； 生根移栽步骤，选取壮苗培养后茎叶生长均勻的芽切下，用吲哚丁酸浸泡后再接入生根培养基进行生根培养，生出符合移栽要求的根后进行炼苗移栽。 Seedlings incubation step, after the shoot elongation medium access seedlings were cultured seedlings; rooting transplanting step, select them, then cultured seedlings sprout leaf growth even cut, soaked with indole butyric acid and then take After the rooting medium was rooting, birth roots were in line with the requirements of hardening transplant transplanting.

[0009] 进一步地，所述诱导增殖步骤中，增殖培养基的成分为：MS+0. Γ2 mg/L 6_苄基嘌呤+0. 01〜2 mg/L吲哚丁酸+0〜5 mg/L的硝酸银，附加3%蔗糖和0. 7%琼脂，ρΗ5· 8。 [0009] Further, the step of inducing proliferation, proliferation medium composition was:.. MS + 0 Γ2 mg / L 6_ benzyladenine +0 01~2 mg / L of IBA + 0~5 mg / L of silver nitrate, an additional 3% sucrose and 0.7% agar, ρΗ5 · 8.

[0010] 进一步地，所述诱导增殖步骤中，培养条件是：温度控制在M〜26°C，光照时间为12 h/d培养20〜50天，光照强度为1600^2000 Ix0 [0010] Further, the induction of proliferation step, the culture conditions are: temperature control in M~26 ° C, photoperiod of 12 h / d culture 20~50 days, the light intensity of 1600 ^ 2000 Ix0

[0011] 进一步地，所述伸长培养步骤中，伸长培养基成分为：MS +0. Γ2 mg/L 6_苄基嘌呤和(Tl mg/L的赤霉素，附加3%蔗糖和0. 7%琼脂，ρΗ5· 8。 [0011] Further, the elongation step of culturing, the elongation medium ingredients:. MS +0 Γ2 mg / L 6_ benzyladenine and (Tl mg / L of GA, an additional 3% sucrose and 0.7% agar, ρΗ5 · 8.

[0012] 进一步地，所述伸长培养步骤中，培养条件是：温度控制在24l6°C，光照时间为12 h/d培养10〜30天，光照强度为1600^2000 Ix0 [0012] Further, the elongation step of culturing, the culture conditions are: temperature control in 24l6 ° C, photoperiod of 12 h / d culture 10~30 days, the light intensity of 1600 ^ 2000 Ix0

[0013] 进一步地，所述壮苗培养步骤中，壮苗培养基中含有：MS+0. Γ2 mg/L 6_苄基嘌呤+0.0广2 mg/L吲哚丁酸+0〜5 mg/L的硝酸银+(TlO %椰汁。 [0013] Further, the seedlings culture step, seedling medium containing:. MS + 0 Γ2 mg / L 6_ benzyladenine +0.0 wide 2 mg / L of IBA + 0~5 mg / L silver nitrate + (TlO% coconut milk.

[0014] 进一步地，所述壮苗培养步骤中，培养条件为：温度控制在M〜26°C，光照时间为12 h/d培养10〜30天，光照强度为1600^2000 Ix0 [0014] Further, the seedlings culturing step, the culture conditions: temperature control in M~26 ° C, photoperiod of 12 h / d culture 10~30 days, the light intensity of 1600 ^ 2000 Ix0

[0015] 进一步地，所述生根移栽步骤中，生根培养基为1/2 MS+0. Γ2 mg/L的吲哚丁酸， 需附加广2 %的蔗糖、0. 3〜0. 7 %的琼脂，调pH值至5. 8。 [0015] Further, the transplanting step rooting, rooting medium was 1/2 MS + 0. Γ2 mg / L IBA for additional wide 2% sucrose, 0. 3~0. 7 % of agar, pH was adjusted to 5.8.

[0016] 进一步地，所述生根移栽步骤中，壮苗培养后茎叶生长均勻的芽的切法为：选取生长均勻的芽在结下广4 mm处平切。 [0016] Further, the rooting transplanting step, even after seedling cultivation leaf growth bud cut law: Choose a uniform bud growth in forged flat cut at 4 mm wide.

[0017] 进一步地，所述生根移栽步骤中，切下的芽在接入生根培养基之前先在0. Γ2 mg/ L的吲哚丁酸中浸泡5〜120 min。 [0017] Further, the rooting transplanting step, bud cut soak 5~120 min at 0. Γ2 mg / L IBA in the rooting medium before access.

[0018] 通过采用上述技术方案，本发明至少具有如下有益效果：本发明中采用再生芽来诱导增殖，可用于小桐子遗传转化后优良品系增殖，为小桐子的人工繁殖、脱毒苗生产、繁殖优良品种开辟高效途径。 [0018] By using the techniques described above, the present invention has at least the following beneficial effects: the present invention is used to induce regeneration bud proliferation, it can be used for Jatropha genetic transformation after fine lines proliferation of artificial propagation of Jatropha, virus-free seedling production, breeding improved varieties open and efficient way.

[0019] 本发明所用的增殖培养基添加有细胞分裂素6-苄基嘌呤和生长素吲哚丁酸，增殖倍数多，平均可达3飞倍，因此，仅需少量的芽就可在短时间内得到大量的材料，为小桐子组培中选育的优良品种的大批量繁殖奠定了基础。 [0019] proliferation medium used in the present invention is added 6-benzyl purine cytokinin and auxin indole butyric acid, more proliferation rate, an average of up to 3 flight times, therefore, only a small number of shoots can be in short time to get a lot of material, large quantities of Jatropha tissue culture varieties bred breeding foundation.

[0020] 本发明的方法中在得到增殖的芽后进行了茎叶伸长培养和壮苗培养，保证了增殖芽都能顺利发育为完整植株。 [0020] The method of the present invention, after obtaining the proliferation buds were cultured stem elongation and seedling cultivation, to ensure that the proliferation of buds can successfully develop into whole plants.

[0021] 本发明的方法是在无菌操作中完成，不受时间和地域的限制，可随时完成。 [0021] The method of the present invention is done in a sterile operation, without time and geographical constraints, can be completed at any time. 具体实施方式 DETAILED DESCRIPTION

[0022] 本发明提供一种小桐子快速繁殖方法，包括以下步骤： [0022] The present invention provides a method for rapid propagation of Jatropha, comprising the steps of:

准备再生芽步骤，以小桐子成熟种子为材料，经培育获得无菌再生芽； 增殖诱导步骤，将再生芽切下，接入增殖培养基进行增殖培养，从而获得增殖的丛芽；伸长培养步骤，将增殖后的丛芽切下，接入伸长培养基进而获得茎叶伸长的均勻的 Prepare regeneration bud steps to Jatropha seeds mature material, obtained by cultivating sterile regeneration bud; proliferation induced by step, the regenerated shoots cut down access to proliferate proliferation culture medium to obtain proliferation of buds; elongation culture steps to proliferate after the buds cut, then get access elongation medium uniform stem elongation

芽； Bud;

壮苗培养步骤，将伸长后的芽接入壮苗培养基进行壮苗培养； 生根移栽步骤，选取壮苗培养后茎叶生长均勻的芽切下接入生根培养基进行生根培养，待培养出符合移栽要求的根后进行炼苗移栽。 Seedlings incubation step, after the shoot elongation medium access seedlings were cultured seedlings; rooting transplanting step, select them, then cultured seedlings sprout leaf growth even cut access were rooting rooting medium, to be After incubation the root will be in line with the requirements of hardening transplant transplanting.

[0023] 其中，准备再生芽步骤Sl主要是为获得无菌再生芽以供后续增殖诱导步骤使用， 采用现有的各种利用种子培育发芽技术均可实现本步骤的目的。 [0023] where regeneration bud ready to step Sl primarily to obtain sterile regeneration bud proliferation-inducing step for subsequent use, the use of various existing Seed germination foster technology can achieve the purpose of this step.

[0024] 增殖诱导步骤S2进一步包括以下工艺： [0024] proliferation-inducing step S2 further includes the following process:

52. 1将分化出的再生芽切下； 52.1 The differentiated cut regenerated shoots;

S2.2将切下的再生芽接入增殖培养基，该增殖培养基是指：MS+0. Γ2 mg/L 6-苄基嘌呤+0.01〜2 mg/L吲哚丁酸+0〜5 mg/L的硝酸银，需附加3 %蔗糖、0.7 %琼脂，pH5.8。 S2.2 The regenerated shoots proliferation medium access cut, the proliferation medium means:. MS + 0 Γ2 mg / L 6- benzyladenine + 0.01~2 mg / L of IBA + 0~5 mg / L of silver nitrate for additional 3% sucrose, 0.7% agar, pH5.8.

[0025] S2. 3培养条件为温度24l6°C，光照时间为12 h/d培养20〜50天，光照强度为1600〜2000 lx。 [0025] S2. 3 culture conditions for the temperature 24l6 ° C, photoperiod of 12 h / d culture 20~50 days, light intensity 1600~2000 lx.

[0026] 伸长培养步骤S3进一步包括以下工艺： [0026] culturing step S3 elongation further includes the following process:

53. 1将增殖后的丛芽切下接入伸长培养基，该伸长培养基为MS +0. Γ2 mg/L 6-苄基嘌呤和(Tl mg/L的赤霉素，需附加3 %蔗糖、0. 7 %琼脂，pH5. 8。 53.1 The proliferated under buds cut access elongation medium, the elongation medium is MS +0. Γ2 mg / L 6- benzyladenine and (Tl mg / L of GA for additional 3% sucrose, 0.7% agar, pH5. 8.

[0027] S3. 2培养条件为温度24l6°C，光照时间为12 h/d培养20〜50天，光照强度为1600〜2000 lx。 [0027] S3. 2 culture conditions for temperature 24l6 ° C, photoperiod of 12 h / d culture 20~50 days, light intensity 1600~2000 lx.

[0028] 壮苗培养步骤S4进一步包括以下工艺： [0028] seedling cultivation step S4 further comprises the following process:

54. 1伸长后的芽切下接入壮苗培养基，该壮苗培养基成分包括：MS+0.广2 mg/L 6-苄基嘌呤+0.0广2 mg/L吲哚丁酸+0〜5 mg/L的硝酸银+(TlO %椰汁; Access medium seedling shoots cut at 54.1 after being stretched, the seedlings medium components include:. MS + 0 Canton 2 mg / L 6- benzyladenine +0.0 wide 2 mg / L indole butyric acid + 0~5 mg / L of silver nitrate + (TlO% coconut milk;

S4.2培养温度控制在24l6°C，光照时间为12 h/d培养10〜30天，光照强度为1600〜2000 lx。 S4.2 culture temperature control in 24l6 ° C, illumination time is 12 h / d culture 10~30 days, light intensity 1600~2000 lx.

[0029] 生根移栽步骤S5中进一步包括以下工艺： [0029] rooting transplanting step S5 further comprises the following process:

55. 1选取茎叶生长均勻的芽切下，切法为：苗在结下广4 mm处平切； S5. 2并在0. Γ2 mg/L的吲哚丁酸中浸泡5〜120 min，接入生根培养基； Select the leaf growth at 55.1 uniform bud cut, cut law: seedlings forged flat cut at 4 mm wide; S5 2 and soaked 5~120 min at 0. Γ2 mg / L indole butyric acid. access rooting medium;

S5.3待培养出符合移栽要求的根后进行炼苗移栽，通常地，当培养出3条以上的根， 且每条根长度为3cm长时即可进行炼苗移栽。 After S5.3 be cultivated roots were in line with the requirements of hardening transplant transplanting, usually, when the train more than three roots, and the length of each root can be transplanted hardening 3cm long.

[0030] 下面结合几个具体的实施例详述本发明的实施方案。 [0030] The following examples combine several specific embodiments of the present invention is described in detail. 需要说明的是，下述实施例是说明性的，不是限定性的，不能以下述实例来限定本发明的保护范围。 It should be noted that the following examples are illustrative and not restrictive, the following examples should not limit the scope of the present invention. 此外，如前所述，采用现有的各种利用种子培育发芽技术均可实现准备再生芽步骤Si的目的，该准备再生芽步骤Sl并非本发明创新的核心所在，因此，在以下各实施例中，将不再特别描述准备再生芽步骤Si，而直接从增殖诱导步骤S2开始阐述。 In addition, as mentioned above, the use of various existing Seed germination foster technology can achieve regeneration bud ready to step Si purpose, the regeneration bud ready to step Sl is not the core of the present invention and innovation, therefore, in the following examples will no longer be specifically described regeneration buds ready to step Si, began to expound directly from the proliferation-inducing step S2.

[0031] 实施例1 [0031] Example 1

本实例以云南元谋野生小桐子种子为材料，样品的总数量及经各处理步骤后的存活数量请参考表1。 The instances in Yuanmou wild Jatropha seeds as material, the total number of samples and the number of surviving after each processing step. See Table 1. 各主要工艺步骤具体如下： Each main process steps as follows:

增殖诱导步骤S2:将分化出的再生芽切下，接入增殖培养基MP8，即MS+1.0 mg/L 6-苄基嘌呤+0. 01mg/L吲哚丁酸+0. 025 mg/L的硝酸银，并附加3%蔗糖和0. 7%琼脂，pH5. 8，温度控制在对！ A proliferation-inducing step S2: Under the differentiated cut regenerated shoots, access proliferation medium MP8, namely MS + 1.0 mg / L 6- benzyladenine +0 01mg / L of IBA +0 025 mg / L.. silver nitrate, and an additional 3% sucrose and 0.7% agar, pH5. 8, temperature control in the right! ：，光照时间为12 h/d培养30天，光照强度为1800 Ix0[0032] 伸长培养步骤S3 ：将增殖后的丛芽切下，接种到伸长培养基SEB3，即MS+0. 3 mg/L 6-苄基嘌呤和0. 25 mg/L的赤霉素，并附加3 %蔗糖和0. 7%琼脂，ρΗ5· 8，诱导茎叶伸长， 培养温度控制在对〜261：，光照时间为12 h/d培养30天，光照强度为160(T2000 Ix0 : Illumination time of 12 h / d for 30 days, the light intensity of 1800 Ix0 [0032] elongation culturing step S3: the lower buds cut growth after inoculation to elongation medium SEB3, namely MS + 0 3. mg / L 6- benzyladenine and 0. 25 mg / L of GA, and an additional 3% sucrose and 0.7% agar, ρΗ5 · 8, induced stem elongation, temperature control of ~261: illumination time of 12 h / d for 30 days, the light intensity of 160 (T2000 Ix0

[0033] 壮苗培养步骤S4 ：伸长后的芽切下接入壮苗培养基MCl，即MS+1. 0 mg/L 6_苄基嘌呤+0.01 mg/L吲哚丁酸+0 mg/L的硝酸银+5 %椰汁，进行壮苗培养，培养温度控制在^°C，光照时间为12 h/d培养18天，光照强度2000 Ix0 [0033] seedling cultivation step S4: access the next cut elongation after the seedlings sprout medium MCl, namely MS + 1 0 mg / L 6_ benzyladenine +0.01 mg / L indole butyric acid +0 mg. / L silver nitrate +5% coconut milk, were cultured seedlings, cultivation temperature is controlled at ^ ° C, illumination time is 12 h / d for 18 days, the light intensity 2000 Ix0

[0034] 生根移栽步骤S5 ：取灭菌的盘，垫上无菌滤纸，加入少量无菌水；挑取长势均勻(对于长势不均勻的芽要再经过壮苗培养后才能进行生根培养）的芽切下，切法为：在芽的结下2 mm处平切，并在0.3 mg/L的吲哚丁酸中浸泡5 min，浸泡过程中尽量只让芽的切口处接触到液体；然后取出芽接种到生根培养基R2中诱导生根，该生根培养基R2成分为： 1/2MS+0. 3 mg/L的吲哚丁酸，并附加1%的蔗糖和0. 35%的琼脂，并调pH值至5. 8。 [0034] rooting transplanting step S5: Take sterilization tray, pad sterile filter paper, add a small amount of sterile water; growing even picked (for uneven after growing seedlings sprout again after rooting culture to culture) of Under buds cut, cut law: forged in the bud at the level of 2 mm cut and soaked in 0.3 mg / L IBA in 5 min, soaked in the process as much as possible just to make the cut at the bud to liquids; and Remove shoots were rooting medium to induce rooting R2, R2 component of the rooting medium:. 1 / 2MS + 0 3 mg / L IBA, and an additional 1% sucrose and 0.35% agar, and adjust the pH to 5.8. 培养条件是：温度控制在M〜26°C，光照时间为12 h/d培养14天，光照强度为1600 Ix0选取已生根至少3条的生根苗，在自然光条件下过度广3星期，逐渐开盖炼苗并移栽至温室。 Culture conditions: temperature control in M~26 ° C, illumination time is 12 h / d for 14 days, the light intensity of 1600 Ix0 select at least three of which has taken root rooted in natural light conditions over wide three weeks, gradually open Cover hardening and transplanted to the greenhouse.

[0035] 实施例2 [0035] Example 2

本实例以云南元谋野生小桐子种子为材料，样品的总数量及经各处理步骤后的存活数量请参考表1。 The instances in Yuanmou wild Jatropha seeds as material, the total number of samples and the number of surviving after each processing step. See Table 1. 各主要工艺步骤具体如下： Each main process steps as follows:

增殖诱导步骤：将分化出的再生芽切下，接入增殖培养基MP16，即MS+2.0 mg/L 6-苄基嘌呤+0. 1 mg/L吲哚丁酸+1 mg/L的硝酸银，并附加3%蔗糖和0. 7%琼脂，pH5. 8，培养温度控制在沈！ A proliferation-inducing steps: Under the differentiated cut regenerated shoots, access proliferation medium MP16, ie MS + 2.0 mg / L 6- benzyladenine +0 1 mg / L indole butyric acid +1 mg / L of nitrate. silver, and an additional 3% sucrose and 0.7% agar, pH5. 8, the culture temperature is controlled sink! ：，光照时间为12 h/d培养35天，光照强度为2000 Ix0 : Illumination time of 12 h / d cultured 35 days, light intensity of 2000 Ix0

[0036] 伸长培养步骤：将增殖后的丛芽切下，接种到伸长培养基SEBlO JPMS +2 mg/L 6-苄基嘌呤和1 mg/L的赤霉素，并附加3 %蔗糖和0.7 %琼脂，pH5. 8，诱导茎叶伸长，培养温度控制在^°C，光照时间为12 h/d培养20天，光照强度为1800 Ix0 [0036] elongation culturing step: cut the lower buds proliferation after inoculation to elongation medium SEBlO JPMS +2 mg / L 6- benzyladenine and 1 mg / L of GA, and an additional 3% sucrose and 0.7% agar, pH5. 8, induced stem elongation, temperature control at ^ ° C, photoperiod of 12 h / d for 20 days, the light intensity of 1800 Ix0

[0037] 壮苗培养步骤：伸长后的芽切下接入壮苗培养基MC2，即改良MS+2. 0 mg/L 6_苄基嘌呤+0.1 mg/L吲哚丁酸+1 mg/L的硝酸银+10 %椰汁，进行壮苗培养；培养温度控制在^°C，光照时间为12 h/d培养14天，光照强度2000 Ix0 [0037] seedling cultivation step: access the next cut elongation after the seedlings sprout medium MC2, namely improved MS + 2 0 mg / L 6_ benzyladenine +0.1 mg / L indole butyric acid +1 mg. / L silver nitrate +10% coconut milk, were seedlings culture; the culture temperature is controlled at ^ ° C, illumination time is 12 h / d for 14 days, light intensity 2000 Ix0

[0038] 生根移栽步骤：取灭菌的盘，垫上无菌滤纸，加入少量无菌水；挑取长势均勻的芽切下，切法为：在芽的结下1 mm处平切，并在1 mg/L的吲哚丁酸中浸泡120 min，浸泡过程中尽量只让芽的切口处接触到液体；然后取出芽接种到生根培养基R2，即1/2MS+1 mg/L的吲哚丁酸，并附加2、的蔗糖和0. 7%的琼脂，并调pH值至5. 8，诱导生根。 [0038] rooting transplanting steps: take the sterilization tray, pad sterile filter paper, add a small amount of sterile water; picked shoots growing even cut, cut law: forged in the bud to 1 mm flat cut, and Soak 120 min at 1 mg / L IBA, the immersion process is only possible to make a cut at the bud to liquids; then, remove buds inoculated into rooting medium R2, namely 1 / 2MS + 1 mg / L of indole IBA, and an additional 2, sucrose and 0.7% agar, and adjust the pH to 5.8, rooting. 培养条件是：温度控制在对〜261：，光照时间为12 h/d培养20天，光照强度为1600 Ix0选取已生根至少3条的生根苗，在自然光条件下过度广3星期，逐渐开盖炼苗并移栽至温室。 Culture conditions: temperature control in the light of the ~261 :, time 12 h / d for 20 days, the light intensity of 1600 Ix0 select at least three of which has taken root rooted in natural light conditions over wide three weeks, gradually opening the lid Hardening and transplanted to the greenhouse.

[0039] 实施例3 [0039] Example 3

本实例以云南元谋野生小桐子种子为材料，样品的总数量及经各处理步骤后的存活数量请参考表1。 The instances in Yuanmou wild Jatropha seeds as material, the total number of samples and the number of surviving after each processing step. See Table 1. 各主要工艺步骤具体如下： Each main process steps as follows:

增殖诱导步骤：将分化出的再生芽切下，接入增殖培养基ΜΡ0，即MS+0. 1 mg/L 6-苄基嘌呤+0. 01 mg/L吲哚丁酸+5 mg/L的硝酸银，并附加3%蔗糖和0. 7%琼脂，ρΗ5· 8，培养温度控制在沈！ A proliferation-inducing steps: Under the differentiated cut regenerated shoots, access proliferation medium ΜΡ0, namely MS + 0 1 mg / L 6- benzyladenine +0 01 mg / L indole butyric acid +5 mg / L.. silver nitrate, and an additional 3% sucrose and 0.7% agar, ρΗ5 · 8, the culture temperature is controlled sink! ：，光照时间为12 h/d培养40天，光照强度为2000 Ix0 : Illumination time of 12 h / d for 40 days, the light intensity of 2000 Ix0

[0040] 伸长培养步骤：将增殖后的丛芽切下，接种到伸长培养基SEBl JPMS +0.1 mg/L 6-苄基嘌呤和0. 5 mg/L的赤霉素，并附加3 %蔗糖和0. 7%琼脂，pH5. 8，诱导茎叶伸长，培养温度控制在M〜26°C，光照时间为12 h/d培养30天，光照强度为2000 Ix0 [0040] elongation culturing step: cut the lower buds proliferation after inoculation to elongation medium SEBl JPMS +0.1 mg / L 6- benzyladenine and 0. 5 mg / L of GA, and an additional 3 % sucrose and 0.7% agar, pH5. 8, induced stem elongation, temperature control in M~26 ° C, photoperiod of 12 h / d for 30 days, the light intensity of 2000 Ix0

[0041] 壮苗培养步骤：伸长后的芽切下接入壮苗培养基MC1，即MS+0. 3 mg/L 6_苄基嘌呤+0.1 mg/L吲哚丁酸+0.5 mg/L的硝酸银+0 %椰汁，进行壮苗培养；培养温度控制在^°C，光照时间为12 h/d培养20天，光照强度2000 Ix0 [0041] seedling cultivation step: access medium seedling shoot MC1 cut down after being stretched, that MS + 0 3 mg / L 6_ benzyladenine +0.1 mg / L of IBA +0.5 mg /. L silver nitrate +0% coconut milk, were strong seedling cultivation; culture temperature is controlled at ^ ° C, photoperiod of 12 h / d for 20 days, the light intensity 2000 Ix0

[0042] 生根移栽步骤：取灭菌的盘，垫上无菌滤纸，加入少量无菌水；挑取长势均勻的芽切下，切法为：在芽的结下4 mm处平切，并在2 mg/L的吲哚丁酸中浸泡60min，浸泡过程中尽量只让芽的切口处接触到液体；然后取出芽接种到生根培养基R2，即1/2 MS+2 mg/L的吲哚丁酸，并附加1. 5%的蔗糖和7%的琼脂，并调pH值至5. 8，诱导生根。 [0042] rooting transplanting steps: take the sterilization tray, pad sterile filter paper, add a small amount of sterile water; picked shoots growing even cut, cut law: in bud forged 4 mm at the flat cut, and 60min soak in 2 mg / L IBA, the soaking process as much as possible so that the bud of the incision only to liquids; then, remove buds inoculated into rooting medium R2, namely 1/2 MS + 2 mg / L of indole IBA, and an additional 1.5% and 7% sucrose agar, and adjust the pH to 5.8, rooting. 其培养的条件是： 温度控制在M〜26°C，光照时间为12 h/d培养18天，光照强度为1600 Ix0选取已生根至少3条的生根苗，在自然光条件下过度广3星期，逐渐开盖炼苗并移栽至温室。 The conditions of its culture are: temperature control in M~26 ° C, illumination time is 12 h / d for 18 days, the light intensity of 1600 Ix0 select at least three of which has taken root rooted in natural light conditions over wide three weeks, openings gradually hardening and transplanting to the greenhouse.

[0043] 表1本发明各实施例中的再生率及增值率 Examples regeneration rate and the added value of each of the embodiments [0043] Table 1 invention

从上表中可以看出，获得的增殖芽数量最低也能达到再生芽数量的3倍以上，仅需少量的芽就可在短时间内得到大量的材料，为小桐子组织培养选育的优良品种的大批量繁殖奠定了基础。 As can be seen from the above table, the minimum number of proliferating shoots obtained can reach more than three times the number regeneration bud, only a small number of buds can get a lot of material in a short time, as Jatropha breeding excellent tissue culture Large quantities of species breeding foundation.